Bottom Line:
We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells.Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer) in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT) cells.This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells.

ABSTRACTCLEC4F, a member of C-type lectin, was first purified from rat liver extract with high binding affinity to fucose, galactose (Gal), N-acetylgalactosamine (GalNAc), and un-sialylated glucosphingolipids with GalNAc or Gal terminus. However, the biological functions of CLEC4F have not been elucidated. To address this question, we examined the expression and distribution of murine CLEC4F, determined its binding specificity by glycan array, and investigated its function using CLEC4F knockout (Clec4f-/-) mice. We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells. In contrast to F4/80, CLEC4F is detectable in fetal livers at embryonic day 11.5 (E11.5) but not in yolk sac, suggesting the expression of CLEC4F is induced as cells migrate from yolk cells to the liver. Even though CLEC4F is not detectable in tissues outside liver, both residential Kupffer cells and infiltrating mononuclear cells surrounding liver abscesses are CLEC4F-positive upon Listeria monocytogenes (L. monocytogenes) infection. While CLEC4F has strong binding to Gal and GalNAc, terminal fucosylation inhibits CLEC4F recognition to several glycans such as Fucosyl GM1, Globo H, Bb3∼4 and other fucosyl-glycans. Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer) in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT) cells. This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells.

pone-0065070-g002: CLEC4F is co-expressed with F4/80 on liver Kupffer cells.(A) CLEC4F and F4/80 immunohistochemistry of parafilm-embedded liver sections from wild-type and Clec4f−/− mice. (B) Double immunofluorescence of CLEC4F and F4/80 in wild-type livers was performed. Nuclei were counterstained with Hoechst 33342. Signals were determined by confocal microscope (magnification 10×63). (C) Coexpression of CLEC4F and F4/80 on Kupffer cells, but not peritoneal macrophages. Cells were double stained with Alexa Fluor 647-conjugated anti-F4/80 and PE-conjugated anti-CLEC4F mAb. Alexa Fluor 647-conjugated rat IgG2b and PE-conjugated mIgG1 were used as isotype controls.

Mentions:
The localization of CLEC4F+ cells was determined by immunohistochemistry. By using tissue array, we found that CLEC4F is only expressed in liver sinusoid cells, but not in other tissues (Figure S2 in File S1). For further confirmation of the binding specificity of anti-CLEC4F mAb, liver sections from wild-type (upper panel, Figure 2A) and Clec4f−/− (lower panel, Figure 2A) mice were used. The worm-like cells located in the lumen of sinusoid were detectable by anti-CLEC4F mAb and anti-F4/80 Ab, respectively (upper panel, Figure 2A), while only anti-F4/80 Ab, but not anti-CLEC4F mAb, could detect Kupffer cells in Clec4f−/− liver section (lower panel, Figure 2A). In Clec4f−/− mice, the numbers and distribution of F4/80+ Kupffer cells are similar to wild-type mice, suggesting that CLEC4F is dispensable for Kupffer cell development.

pone-0065070-g002: CLEC4F is co-expressed with F4/80 on liver Kupffer cells.(A) CLEC4F and F4/80 immunohistochemistry of parafilm-embedded liver sections from wild-type and Clec4f−/− mice. (B) Double immunofluorescence of CLEC4F and F4/80 in wild-type livers was performed. Nuclei were counterstained with Hoechst 33342. Signals were determined by confocal microscope (magnification 10×63). (C) Coexpression of CLEC4F and F4/80 on Kupffer cells, but not peritoneal macrophages. Cells were double stained with Alexa Fluor 647-conjugated anti-F4/80 and PE-conjugated anti-CLEC4F mAb. Alexa Fluor 647-conjugated rat IgG2b and PE-conjugated mIgG1 were used as isotype controls.

Mentions:
The localization of CLEC4F+ cells was determined by immunohistochemistry. By using tissue array, we found that CLEC4F is only expressed in liver sinusoid cells, but not in other tissues (Figure S2 in File S1). For further confirmation of the binding specificity of anti-CLEC4F mAb, liver sections from wild-type (upper panel, Figure 2A) and Clec4f−/− (lower panel, Figure 2A) mice were used. The worm-like cells located in the lumen of sinusoid were detectable by anti-CLEC4F mAb and anti-F4/80 Ab, respectively (upper panel, Figure 2A), while only anti-F4/80 Ab, but not anti-CLEC4F mAb, could detect Kupffer cells in Clec4f−/− liver section (lower panel, Figure 2A). In Clec4f−/− mice, the numbers and distribution of F4/80+ Kupffer cells are similar to wild-type mice, suggesting that CLEC4F is dispensable for Kupffer cell development.

Bottom Line:
We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells.Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer) in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT) cells.This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells.

ABSTRACTCLEC4F, a member of C-type lectin, was first purified from rat liver extract with high binding affinity to fucose, galactose (Gal), N-acetylgalactosamine (GalNAc), and un-sialylated glucosphingolipids with GalNAc or Gal terminus. However, the biological functions of CLEC4F have not been elucidated. To address this question, we examined the expression and distribution of murine CLEC4F, determined its binding specificity by glycan array, and investigated its function using CLEC4F knockout (Clec4f-/-) mice. We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells. In contrast to F4/80, CLEC4F is detectable in fetal livers at embryonic day 11.5 (E11.5) but not in yolk sac, suggesting the expression of CLEC4F is induced as cells migrate from yolk cells to the liver. Even though CLEC4F is not detectable in tissues outside liver, both residential Kupffer cells and infiltrating mononuclear cells surrounding liver abscesses are CLEC4F-positive upon Listeria monocytogenes (L. monocytogenes) infection. While CLEC4F has strong binding to Gal and GalNAc, terminal fucosylation inhibits CLEC4F recognition to several glycans such as Fucosyl GM1, Globo H, Bb3∼4 and other fucosyl-glycans. Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer) in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT) cells. This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells.